Abstract: Diclofenac (DCF) is one of the widely used non-steroidal anti-inflammatory drugs. With the increased production and consumption, DCF is frequently detected in the environment. DCF can be enriched in organisms and has potential toxicity to several organisms, causing much concern. The accuracy determination of DCF is the basis for other studies. In a previous study, pretreatment methods were always used to eliminate matrix effects during the measurement of DCF, but how the sample matrix influences DCF measurement was not studied in details. In order to meet the need of field sample measurement and laboratory research, the effects of common anions SO₄^2-, Cl^- and NO₃^-, solution pH, and mobile phase constitutions on the determination of DCF using High Performance Liquid Chromatography with the UV-Vis detector (HPLC-UV) were investigated. The results show that SO₄^2- and Cl^- had no effect on the maximum absorption wavelength of DCF (277 nm), but NO₃^- changed the maximum absorption wavelength of DCF (277 nm) and resulted in the red shift phenomenon. Under the same condition, the measured concentration of DCF by HPLC in acidic medium (pH < 5) was lower than that in an alkaline medium. Comparing with the peak area of DCF in alkaline medium (pH=7.26), the peak area of DCF in acidic medium (pH=2.01) decreased by 73.14%. Therefore, determination of DCF under alkaline conditions has greater accuracy.